Single-molecule Fluorescence Approaches Research Articles

Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements.

The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Despite its biological importance, the mechanism by which p53 binds REs remains unclear. To address this, we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of full-length human p53 to five native REs in real time under equilibrium conditions. Our approach enabled us to quantify the oligomeric state of DNA-bound p53. We found little evidence that dimer/DNA complexes form as intermediates en route to binding or dissociation of p53 tetramer/DNA complexes. Interestingly, however, at some REs dimers can rapidly exchange from tetramer/DNA complexes. Real time kinetic measurements enabled us to determine rate constants for association and dissociation at all five REs, which revealed two kinetically distinct populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing that the more favorable binding sequences form the least kinetically stable complexes. Together our single molecule measurements provide new insight into mechanisms by which tetrameric p53 forms complexes on different native REs.

Single-molecule approaches for DNA damage detection and repair: A focus on Repair Assisted Damage Detection (RADD)

The human genome is continually exposed to various stressors, which can result in DNA damage, mutations, and diseases. Among the different types of DNA damage, single-strand lesions are commonly induced by external stressors and metabolic processes. Accurate detection and quantification of DNA damage are crucial for understanding repair mechanisms, assessing environmental impacts, and evaluating response to therapy. However, traditional techniques have limitations in sensitivity and the ability to detect multiple types of damage. In recent years, single-molecule fluorescence approaches have emerged as powerful tools for precisely localizing and quantifying DNA damage. Repair Assisted Damage Detection (RADD) is a single-molecule technique that employs specific repair enzymes to excise damaged bases and incorporates fluorescently labeled nucleotides to visualize the damage. This technique provides valuable insights into repair efficiency and sequence-specific damage. In this review, we discuss the principles and applications of RADD assays, highlighting their potential for enhancing our understanding of DNA damage and repair processes.

Methods to investigate nucleosome structure and dynamics with single-molecule FRET.

The nucleosome is the fundamental building block of chromatin. Changes taking place at the nucleosome level are the molecular basis of chromatin transactions with various enzymes and factors. These changes are directly and indirectly regulated by chromatin modifications such as DNA methylation and histone post-translational modifications including acetylation, methylation, and ubiquitylation. Nucleosomal changes are often stochastic, unsynchronized, and heterogeneous, making it very difficult to monitor with traditional ensemble averaging methods. Diverse single-molecule fluorescence approaches have been employed to investigate the structure and structural changes of the nucleosome in the context of its interactions with various enzymes such as RNA Polymerase II, histone chaperones, transcription factors, and chromatin remodelers. We utilize diverse single-molecule fluorescence methods to study the nucleosomal changes accompanying these processes, elucidate the kinetics of these processes, and eventually learn the implications of various chromatin modifications in directly regulating these processes. The methods include two- and three-color single-molecule fluorescence resonance energy transfer (FRET), single-molecule fluorescence correlation spectroscopy, and fluorescence (co-)localization. Here we report the details of the two- and three-color single-molecule FRET methods we currently use. This report will help researchers design their single-molecule FRET approaches to investigating chromatin regulation at the nucleosome level.

Assembly of Synaptic Protein-DNA Complexes: Critical Role of Non-Specific Interactions.

The synaptic protein-DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site-search process for synaptic DNA-protein systems. To investigate the molecular mechanism behind these site-search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific-specific (synaptic), non-specific-non-specific (non-specific), and specific-non-specific (pre-synaptic) SfiI-DNA states. Unexpectedly, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein-DNA complexes discovered in the time-lapse AFM experiments.

Characterisation of Amyloid Aggregation and Inhibition by Diffusion-Based Single-Molecule Fluorescence Techniques

Protein amyloid aggregation has been associated with more than 50 human disorders, including the most common neurodegenerative disorders Alzheimer’s and Parkinson’s disease. Interfering with this process is considered as a promising therapeutic strategy for these diseases. Our understanding of the process of amyloid aggregation and its role in disease has typically been limited by the use of ensemble-based biochemical and biophysical techniques, owing to the intrinsic heterogeneity and complexity of the process. Single-molecule techniques, and particularly diffusion-based single-molecule fluorescence approaches, have been instrumental to obtain meaningful information on the dynamic nature of the fibril-forming process, as well as the characterisation of the heterogeneity of the amyloid aggregates and the understanding of the molecular basis of inhibition of a number of molecules with therapeutic interest. In this article, we reviewed some recent contributions on the characterisation of the amyloid aggregation process, the identification of distinct structural groups of aggregates in homotypic or heterotypic aggregation, as well as on the study of the interaction of amyloid aggregates with other molecules, allowing the estimation of the binding sites, affinities, and avidities as examples of the type of relevant information we can obtain about these processes using these techniques.

MRNA- and factor-driven dynamic variability controls eIF4F-cap recognition for translation initiation.

mRNA 5′ cap recognition by eIF4F is a key element of eukaryotic translational control. Kinetic differences in eIF4F–mRNA interactions have long been proposed to mediate translation-efficiency differences between mRNAs, and recent transcriptome-wide studies have revealed significant heterogeneity in eIF4F engagement with differentially-translated mRNAs. However, detailed kinetic information exists only for eIF4F interactions with short model RNAs. We developed and applied single-molecule fluorescence approaches to directly observe real-time Saccharomyces cerevisiae eIF4F subunit interactions with full-length polyadenylated mRNAs. We found that eIF4E–mRNA association rates linearly anticorrelate with mRNA length. eIF4G–mRNA interaction accelerates eIF4E–mRNA association in proportion to mRNA length, as does an eIF4F-independent activity of eIF4A, though cap-proximal secondary structure still plays an important role in defining the final association rates. eIF4F–mRNA interactions remained dominated by effects of eIF4G, but were modulated to different extents for different mRNAs by the presence of eIF4A and ATP. We also found that eIF4A-catalyzed ATP hydrolysis ejects eIF4E, and likely eIF4E•eIF4G from the mRNA after initial eIF4F•mRNA complex formation, suggesting a mechanism to prepare the mRNA 5′ end for ribosome recruitment. Our results support a role for mRNA-specific, factor-driven eIF4F association rates in kinetically controlling translation.

Modular single-molecule microscopy: new hardware, software, protocols and controls - the smfbox evolved

Single-molecule fluorescence approaches have given us direct insight into the heterogeneity and dynamics of many important biomolecular systems. However, due to the complexity of most available hardware and software, these methods remain inaccessible to many scientists. Here we present our approach to bring these powerful methods to the widest possible userbase, establishing a modular system to serve as a platform for a range of single-molecule techniques, to include multicolour confocal and TIRF smFRET, fluorescence (cross) correlation spectroscopy and magnetic tweezers.

Single‐Molecule Dynamics of Eukaryotic Translation Initiation

Translation initiation is a critical control point for eukaryotic gene expression. The molecular mechanism of initiation requires a highly dynamic and exquisitely coordinated interplay of mRNA with the translation machinery. Recognition of the mRNA 5ʹ cap structure by initiation factor eIF4F is a key early initiation step, and plays important roles in translational control. We have developed single-molecule fluorescence approaches to directly observe eIF4F–mRNA interaction in real time, both on individual mRNAs and at transcriptome scale with single-molecule resolution. Our data reveal order-of-magnitude variability across mRNAs for interaction of the cap-binding eIF4F subunit, eIF4E, and provide insights into how the eIF4F subunits eIF4G and eIF4A dynamically modulate cap recognition. The data implicate features throughout the mRNA as controlling cap recognition at the 5ʹ end, including a relationship between coding-sequence length and the accessibility of the cap for recognition. We have also developed a single-molecule assay to directly observe the initiation steps that follow eIF4F–mRNA recognition, i.e. 43S ribosomal pre-initiation complex recruitment and pre-initiation complex scanning to locate the start codon. Taken together, our results evoke a multi-layered, mRNA-centric mechanism to kinetically control translation initiation and confer differential sensitivity to distinct regulatory mechanisms.

A translational riboswitch coordinates nascent transcription-translation coupling.

Bacterial messenger RNA (mRNA) synthesis by RNA polymerase (RNAP) and first-round translation by the ribosome are often coupled to regulate gene expression, yet how coupling is established and maintained is ill understood. Here, we develop biochemical and single-molecule fluorescence approaches to probe the dynamics of RNAP-ribosome interactions on an mRNA with a translational preQ1-sensing riboswitch in its 5' untranslated region. Binding of preQ1 leads to the occlusion of the ribosome binding site (RBS), inhibiting translation initiation. We demonstrate that RNAP poised within the mRNA leader region promotes ribosomal 30S subunit binding, antagonizing preQ1-induced RBS occlusion, and that the RNAP-30S bridging transcription factors NusG and RfaH distinctly enhance 30S recruitment and retention, respectively. We further find that, while 30S-mRNA interaction significantly impedes RNAP in the absence of translation, an actively translating ribosome promotes productive transcription. A model emerges wherein mRNA structure and transcription factors coordinate to dynamically modulate the efficiency of transcription-translation coupling.

Single-Molecular Catalysis Identifying Activation Energy of the Intermediate Product and Rate-Limiting Step in Plasmonic Photocatalysis.

Plasmon-mediated photocatalysis provides a novel strategy for harvesting solar energy. Identification of the rate-determining step and its activation energy in plasmon-mediated photocatalysis plays critical roles for understanding the contribution of hot carriers, which facilitates rational designation of catalysts with integrated high photochemical conversion efficiency and catalytic performance. However, it remains a challenge due to a lack of research tools with spatiotemporal resolution that are capable of capturing intermediates. In this work, we used a single-molecule fluorescence approach to investigate a localized surface plasmon resonance (LSPR)-enhanced photocatalytic reaction with subturnover resolution. By introducing variable temperature as an independent parameter in plasmonic photocatalysis, the activation energies of tandem reaction steps, including intermediate generation, product generation, and product desorption, were clearly differentiated, and intermediate generation was found to be the rate-limiting step. Remarkably, the cause of the plasmon-enhanced catalysis performance was found to be its ability of lowering the activation energy of intermediate generation. This study gives new insight into the photochemical energy conversion pathways in plasmon-enhanced photocatalysis and sheds light on designing high-performance plasmonic catalysts.

Prp22 Remodeling of pre-mRNA during the P-to-ILS Spliceosome Transition

The spliceosome is a eukaryotic multi-megadalton macromolecular machine that catalyzes the removal of introns from newly transcribed precursor messenger RNA (pre-mRNA) and ligation of exons to form messenger RNA (mRNA). Instead of existing as a pre-assembled complex, the five small nuclear ribonuclear protein (snRNP) complexes that make up the spliceosome gradually assemble onto each pre-mRNA substrate with each round of splicing. Once the catalytic core of the enzyme is formed, the spliceosome transitions through a series of eight complexes to precisely juxtapose splice sites. The ATP- or GTP- dependent helicase activity of seven essential DExD/H-box helicases is essential to complete each splicing cycle. Further, the kinetics of each spliceosomal rearrangement, and the proteins that tune them, govern splicing fidelity and specificity. Yet despite large amounts of splicing structural and biochemical data, the precise roles of the essential DExD/H-box helicases and the mechanisms by which they transition the spliceosome through the catalytic stage of the cycle remain unclear. To help bridge this knowledge gap, we are developing a single molecule fluorescence approach, guided by recent structural work, to investigate spliceosome complex transitions. Our assay aims to probe the role of the DEAH-box ATP-dependent helicase Prp22 during the post-spliceosome to intron-lariat spliceosome (P-to-ILS) transition. While many proteins have been studied individually at the single-molecule level, the study of elaborate macromolecular machines has been far less common due to challenges associated with purification and data analysis. This work therefore represents a substantial step toward the application of single-molecule spectroscopy to elucidate the molecular mechanisms of complex biological machines.

The C-terminal tail of the yeast mitochondrial transcription factor Mtf1 coordinates template strand alignment, DNA scrunching and timely transition into elongation.

Mitochondrial RNA polymerases depend on initiation factors, such as TFB2M in humans and Mtf1 in yeast Saccharomyces cerevisiae, for promoter-specific transcription. These factors drive the melting of promoter DNA, but how they support RNA priming and growth was not understood. We show that the flexible C-terminal tails of Mtf1 and TFB2M play a crucial role in RNA priming by aiding template strand alignment in the active site for high-affinity binding of the initiating nucleotides. Using single-molecule fluorescence approaches, we show that the Mtf1 C-tail promotes RNA growth during initiation by stabilizing the scrunched DNA conformation. Additionally, due to its location in the path of the nascent RNA, the C-tail of Mtf1 serves as a sensor of the RNA–DNA hybrid length. Initially, steric clashes of the Mtf1 C-tail with short RNA–DNA hybrids cause abortive synthesis but clashes with longer RNA-DNA trigger conformational changes for the timely release of the promoter DNA to commence the transition into elongation. The remarkable similarities in the functions of the C-tail and σ3.2 finger of the bacterial factor suggest mechanistic convergence of a flexible element in the transcription initiation factor that engages the DNA template for RNA priming and growth and disengages when needed to generate the elongation complex.

Protein unties the pseudoknot: S1-mediated unfolding of RNA higher order structure.

Ribosomal protein S1 plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5′ UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of S1 is a central feature of its function. While sequence determinants of S1 affinity and many elements of the interactions of S1 with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of S1 with the well-characterized H-type pseudoknot of a class-I translational preQ1 riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ1, guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that S1 preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of S1 to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of S1-chaperoned unfolding of structured RNAs.

Physical Chemistry of Epigenetics: Single-Molecule Investigations.

The nucleosome is the fundamental building block of the eukaryotic genome, composed of an ∼147 base-pair DNA fragment wrapping around an octameric histone protein core. DNA and histone proteins are targets of enzymatic chemical modifications that serve as signals for gene regulation. These modifications are often referred to as epigenetic modifications that govern gene activities without altering the DNA sequence. Although the term epigenetics initially required inheritability, it now frequently includes noninherited histone modifications associated with gene regulation. Important epigenetic modifications for healthy cell growth and proliferation include DNA methylation, histone acetylation, methylation, phosphorylation, ubiquitination, and SUMOylation (SUMO = Small Ubiquitin-like Modifier). Our research focuses on the biophysical roles of these modifications in altering the structure and structural dynamics of the nucleosome and their implications in gene regulation mechanisms. As the changes are subtle and complex, we employ various single-molecule fluorescence approaches for their investigations. Our investigations revealed that these modifications induce changes in the structure and structural dynamics of the nucleosome and their thermodynamic and kinetic stabilities. We also suggested the implications of these changes in gene regulation mechanisms that are the foci of our current and future research.

PI-3 Single molecule fluorescence approaches to probe the binding of soluble-CD4 to native-like HIV-1 SOSIP trimers

Several lines of evidence suggest that HIV-1 envelope (Env) on virions transition through a series of conformations in solution as well as when they engage CD4 molecules during viral attachment. Moreover, the flexibility and antigenicity of trimers changes as they progressively become saturated with CD4 molecules. HIV-1 Env can potentially engage up to 3 independent CD4 molecules for efficient Env triggering in the virial fusion. However, it is still unknown if one, 2 or 3 CD4 receptor molecules are required for this process. To date, the ensemble techniques and indirect biological assays have been unable to address this question quantitatively. Here, we employed a unique approach utilizing the single molecule detection (SMD) analyses to determine quantitatively the number of sCD4 molecules bound to HIV-1 Env and capture the intermediate interaction states that may be missed by the ensemble methods. A key step in direct counting of single molecules is to ensure single fluorophore labeling of the target molecule. For this purpose, we constructed a C-terminus SNAP-tagged human sCD4 fusion protein and fluorescently labeled it with a single Alexa 488 fluorophore (sCD4-SNAP-A488). This approach was devoid of any genetic or external perturbation to the SOSIP.664 trimers. Our results by SMD confirm that up to 3 sCD4-SNAP-A488 molecules bind a single SOSIP trimer and provide insight into trimer occupancy shifts over time. The experimental data reveals that Env is intrinsically dynamic, transitioning between 3 distinct stoichiometries (1-, 2- and 3-sCD4 bound states). Real-time single molecule association kinetic experiments divulge different states of trimer triggering and stoichiometry of the complexes. We have developed an algorithm for automated analyses of our data to infer the stoichiometry in real-time. Importantly, our single-molecule based association kinetic analysis indicates that assemblies of trimer with 1 or 3 sCD4 molecules are more stable whereas binding of 2 sCD4s seems transitional. Our findings are consistent with structural information available for conformations of HIV-1 Env in its ligand-free, prefusion state and in the CD4-bound states and provide new critical insights into the sCD4 interaction with the Env for better understanding of the complex biology of HIV-1 entry into cells.

Regulation of UvrD Helicase Activity by MutL

Escherichia coli UvrD is a superfamily 1 helicase/translocase involved in multiple DNA metabolic processes including methyl-directed mismatch DNA repair. Although a UvrD monomer can translocate along single-stranded DNA, a UvrD dimer is needed for processive helicase activity in vitro. E. coli MutL, a regulatory protein involved in methyl-directed mismatch repair, stimulates UvrD helicase activity; however, the mechanism is not well understood. Using single-molecule fluorescence and ensemble approaches, we find that a single MutL dimer can activate latent UvrD monomer helicase activity. However, we also find that MutL stimulates UvrD dimer helicase activity. We further find that MutL enhances the DNA-unwinding processivity of UvrD. Hence, MutL acts as a processivity factor by binding to and presumably moving along with UvrD to facilitate DNA unwinding.

Regulation of UvrD Helicase Activity by MutL

Escherichia coli UvrD is a superfamily 1 helicase/translocase involved in multiple DNA metabolic processes including methyl-directed mismatch DNA repair. Although a UvrD monomer can translocate along single-stranded DNA, a UvrD dimer is needed for processive helicase activity in vitro. E. coli MutL, a regulatory protein involved in methyl-directed mismatch repair, stimulates UvrD helicase activity; however, the mechanism is not well understood. Using single-molecule fluorescence and ensemble approaches, we find that a single MutL dimer can activate latent UvrD monomer helicase activity. However, we also find that MutL stimulates UvrD dimer helicase activity. We further find that MutL enhances the DNA-unwinding processivity of UvrD. Hence, MutL acts as a processivity factor by binding to and presumably moving along with UvrD to facilitate DNA unwinding.

High-Speed Magnetic Tweezers Studies of the E. coli Replisome

The replisome is the nucleoprotein assembly responsible for replicating DNA prior to cell division. In E. coli, it comprises roughly a dozen proteins, including DNA polymerase III, DnaB helicase, the beta processivity clamp (β2), and the clamp loader complex. Through a complex hierarchy of dynamic interactions of varying strength, the E. coli replisome can incorporate nearly one thousand nucleotides per second with extremely high fidelity. As such, it represents an ideal model molecular motor, not to mention a potential and heretofore unexploited antibiotic target. In order to study the dynamic behavior of single active replisomes, we employ a magnetic tweezers (MT) assay incorporating a high-speed (several kHz) camera. DNA incorporating a hairpin is tethered between a microscope coverslip and a magnetic bead under applied force. Nucleotide incorporation changes the bead height, allowing us to follow replication with higher spatiotemporal resolution than current single-molecule fluorescence approaches allow. We have previously used this approach to measure rates and identify transient pausing in phage polymerases and present our recent results from the E. coli replisome here.

Mechanistic Insight into the Initiation of Repeat-Associated Nonaug Translation

The predominant genetic cause of amyotrophic lateral sclerosis (ALS) is a hexanucleotide repeat expansion in the intron of C9orf72. Repeat-associated nonAUG (RAN) translation occurs in all six reading frames of the GGGGCC nucleotide expansion tract resulting in the synthesis of neurotoxic dipeptide repeat proteins.We have used bulk biochemical techniques and developed a human translation system for single-molecule fluorescence approaches to elucidate the mechanism of RAN translation initiation.

Large domain movements upon UvrD dimerization and helicase activation

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.